This invention relates to nucleic acid and amino acid sequences of a calcium binding protein and to the use of these sequences in the diagnosis, treatment, and prevention of cancer, reproductive disorders, immune disorders, and developmental disorders.
In nearly all eukaryotic cells, calcium (Ca2+) functions as an intracellular signaling molecule in diverse cellular processes including cell proliferation, neurotransmitter secretion, glycogen metabolism, and skeletal muscle contraction. Within a resting cell, the concentration of Ca2+ in the cytosol is extremely low,  less than 10xe2x88x927 M. However, when the cell is stimulated by an external signal, such as a neural impulse or a growth factor, the cytosolic concentration of Ca2+ increases by about 50-fold. This influx of Ca2+ is caused by the opening of plasma membrane Ca2+ channels and the release of Ca2+ from intracellular stores such as the endoplasmic reticulum. Ca2+ directly activates regulatory enzymes, such as protein kinase C, which trigger signal transduction pathways. Ca2+ also binds to specific Ca2+-binding proteins (CBPs) such as calmodulin (CaM) which then activate multiple target proteins including enzymes, membrane transport pumps, and ion channels. CaM is the most widely distributed and the most common mediator of calcium effects and appears to be the primary sensor of Ca2+ changes in eukaryotic cells. The binding of Ca2+ to CaM induces marked conformational changes in the protein permitting interaction with, and regulation of over 100 different proteins. CaM interactions are involved in a multitude of cellular processes including but not limited to, gene regulation, DNA synthesis, cell cycle progression, mitosis, cytokinesis, cytoskeletal organization, muscle contraction, signal transduction, ion homeostasis, exocytosis, and metabolic regulation (Celio, M. R. et al. (1996) Guidebook to Calcium-binding Protein, Oxford University Press, Oxford, UK, pp. 15-20).
A novel mouse gene named MO25, expressed during early stages of development, has recently been identified and is believed to encode a CBP. The Drosophila equivalent of MO25, DMO25, encodes a polypeptide of 339 amino acid residues with a calculated molecular mass of 39.3 kDa. The novel CBP was found to be conserved among Drosophila, mouse, and yeast. In particular, the carboxy-terminal region of the protein is highly conserved among these species. A homology search revealed that the amino acid sequence of MO25 and DMO25 is similar to a protein encoded in an open reading frame near the calcineurin B subunit gene on chromosome XI in Saccharomyces cerevisiae. Calcineurin B is the small Ca2+-binding regulatory subunit of calcineurin, a CaM-regulated protein phosphatase. The conservation of the MO25 and DMO25 gene structure among species and the wide tissue expression profile indicates that the function of the gene is likely to be fundamental in many cell types as well as during development (Nozaki, M. et al. (1996) DNA Cell Biol. 15:505-509; and Miyamoto, H. et al. (1993) Mol. Reprod. Dev. 34:1-7).
CBPs are implicated in a variety of disorders. For example, calcineurin is found in the cells of all eukaryotes ranging from yeast to mammals. Calcineurin is a target for inhibition by the immunosuppressive agents cyclosporin A and FK506 emphasizing its importance in immune disorders (Kissinger, C. R. et al. (1995) Nature 378:641-644). Calcineurin also plays a critical role in transcriptional regulation and growth control in T-lymphocytes (Wang, M. G. et al. (1996) Cytogenet. Cell Genet. 72:236-241). Additionally, levels of CaM are increased several-fold in tumors and tumor-derived cell lines for various types of cancer (Rasmussen, C. D. and Means, A. R. (1989) Trends in Neuroscience 12:433-438). Calcium binding S100xcex2 is another example of a CBP involved in a variety of disorders. S100xcex2 contains an EF-hand motif and is abundantly expressed in the nervous system. S100xcex2 levels are increased in the blood and cerebrospinal fluid of patients with neurological injury resulting from cerebral infarction, transient ischemic attacks, hemorrhagia, head trauma, and Down""s syndrome. Furthermore, S100xcex2 and other neural-specific CBPs may also protect against neurodegenerative disorders, such as Alzheimer""s, Parkinson""s, and Huntington""s diseases.
The discovery of a new calcium binding protein and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of cancer, reproductive disorders, immune disorders, and developmental disorders.
The invention is based on the discovery of a new human calcium binding protein. (hCBP), the polynucleotides encoding hCBP, and the use of these compositions for the diagnosis, treatment, or prevention of cancer, reproductive disorders, immune disorders, and developmental disorders.
The invention features a substantially purified polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention further provides a substantially purified variant having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also includes an isolated and purified polynucleotide variant having at least 70% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention further provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides an isolated and purified polynucleo comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2, and an isolated and purified polynucleotide variant having at least 70% polynucleotide sequence identity to the polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2. The invention also provides an isolated and purified polynucleotide having a sequence complementary to the polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2.
The invention also provides a method for detecting a polynucleotide in a sample containing nucleic acids, the method comprising the steps of (a) hybridizing the complement of the polynucleotide sequence to at least one of the polynucleotides of the sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide in the sample. In one aspect, the method further comprises amplifying the polynucleotide prior to hybridization.
The invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. In another aspect, the expression vector is contained within a host cell.
The invention also provides a method for producing a polypeptide, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as a purified agonist and a purified antagonist of the polypeptide.
The invention also provides a method for treating or preventing a disorder associated with decreased expression or activity of hCBP, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, in conjunction with a suitable pharmaceutical carrier.
The invention also provides a method for treating or preventing a disorder associated with increased expression or activity of hCBP, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.